Polyethylene glycol (PEG) is a polymer having the structure H(O—CH2—CH2)n—OH. It is generally synthesized by the ring-opening polymerization of ethylene oxide. PEGs of different molecular weight have previously been used in a number of biomedical applications, including processes known as “pegylation,” in which PEG is attached to a protein to extend its activity.
To effect covalent attachment of polyethylene glycol to a protein, the hydroxyl end-groups of the polymer need to be converted to reactive functional groups. This process is frequently referred to as “activation” and the product is called “activated PEG.” Following activation, the reactive functional end-groups of the activated PEG can then react with various functional groups on a variety of molecules, e.g., proteins.
Several chemical procedures have been developed to prepare activated PEGs. For example, PEGs have been successfully activated by reaction with 1,1-carbonyl-di-imidazole, cyanuric chloride, tresyl chloride, 2,4,5-trichlorophenyl chloroformate, 4-nitrophenyl chloroformate, and various N-hydroxy-succinimide derivatives, as well as by the Moffatt-Swern reaction. See Beauchamp et al., Anal. Biochem., 131: 25 (1983); Nashimura et al., Life Sci., 33: 1467 (1983); Delgado et al., Appl. Biochem., 12: 119 (1990); Wirth et al., Bioorg. Chem., 19: 133, (1991); Veronese et al., Biochem. Biotechnol., 11: 141 (1985); Sartore et al., Biochem. Biotechnol., 27: 45 (1991); Anderson et al., J. Immunol. Methods, 109: 37 (1988); and Zalipsky et al., J. Bioact. Compat. Polym., 5: 227 (1990).
The activation of PEGs with 4-nitrophenyl chloroformate to generate PEG-dinitrophenyl carbonates has been described by Fortier and Laliberte. See Fortier et al., Biotech. Appl. Biochem., 17: 115 (1993). The reaction was carried out in acetonitrile containing triethylamine (TEA) for 5 hours at 60° C. To keep an anhydrous environment during the reaction, it was necessary to use a cumbersome Soxhlet extraction system.
International Publication No. WO 03/018665 describes an alternative method for preparing activated PEGs. The method involves a reaction carried out at room temperature using an aprotic solvent, such as methylene chloride (CH2Cl2), in the presence of a catalyst, such as dimethylaminopyridine (DMAP). The use of solvents in both the reaction itself and the subsequent extraction steps to remove by-products, such as the toxic hydrochloric acid salt of DMAP, increases the cost of the final product.
Thus there is a need to develop an efficient, low-cost, and environmentally friendly method for preparing activated PEGs that will minimize or eliminate the use of solvents, catalysts, toxic compounds, cumbersome equipment, and/or purification steps.